Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

BACKGROUND
Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius.


METHODS
This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively.


RESULTS
About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method.


CONCLUSION
Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.


Introduction
arasitic infection including protozoa and helminthes in rodents are of special interest because of the role of rodents as reservoirs of many important parasites of man (1). All rodents are susceptible to protozoan infection. Some of these protozoa may be zoonotic. Although Giardia muris has a limited host and its transmission to human from laboratory rodents has not been reported, care should be taken with Giardia and Cryptosporidium spp. (2,3).
A form of Giardia muris has been observed in the golden hamster, mice, and rats. Infection is usually subclinical but the animal's exhibit weight loss, hair bristling, bloating with meteorism (4).
Trichomonas muris is common parasite that detected in rodents, as mice. Its pathogenesis in mice is unclear. Although T. muris suspected to be non-pathogenic (5), diarrhea and anorexia have been reported as sign of T. muris infection (6).
Although helminthic fauna and Leishmania infection in rodents as a host of visceral Leishmaniosis in Meshkin Shahr district were reported (9,10,11), there is no data on protozoa infection in this area.
This study aimed to determine frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Musmus culus and, Cricetulus migratorius (gray hamster) from Meshkin Shahr district, northwestern Iran.

Materials and Methods
Meshkin Shahr is located in the northwest of Iran in Azerbaijan, It covers an area of ap-proximately1530 km 2 and its population is estimated to be 237585, among whom 29.7% are settled in urban areas and 70.3% live in 323 rural areas. It is the nearest city to the Sabalan high mountain. The weather of this city and the district of Meshkin Shahr is moderate mountainous (12) (Fig.1 All of suspected cases were stained with trichrome staining method (14). The slide was mounted using Canada balsam and observed under 1000x magnification. Detection of intestinal protozoa was based on morphological characteristic of specific protozoa. Analysis was performed using Excel 2007. Formalinether concentration method carried out and smears were prepared from pellet of all samples. The slides were dried at room temperature for 5 min after methanol fixation all of samples were stained with modified acid-fast staining method (15), finally all of slides were observed with under light microscope 1000x magnification. Samples smear were prepared and after drying and methanol fixation, aniline blue staining method carried out according to Ryan method (16). All of samples were observed with 1000x objective and evaluate for detecting microspore spores.
Microspora spores were identified in 63(31.03%) of all samples were stained by the aniline blue staining method. In aniline blue staining method was used for detecting Microspora, ovoid, transluminant spores were observed with 0.7-1.2 µm size. The spores had a belt-like strip in the middle or at the end of body. Trophozoite of E. muris and T. muris stained with trichrome staining method with1000x magnification are shown in (Fig. 2, A) and an unsporulated oocyst of coccidia in a wet mount sample with 400x magnification are shown in (Fig. 2, B) The result of coccidia cultivation in dichromate potassium 2.5% is shown in Fig. 2, C. In each oocyst four sporocysts and in each sporocyst two sporozoites exist, and finally Eimeria spp. diagnosis was made.
The result of acid-fast staining method showed partial acid-fast positive cases with the size of almost 4 µm, in a sample that belongs to a male M. persicus and finally, Cryptosporidium spp diagnosis was made (Fig. 2, D).

Discussion
This study was conducted to determine the prevalence of intestinal protozoa infection of rodents from Meshkin Shahr district, northwestern Iran.
In general, 121 (59.3%) of rodents were infected with protozoa. The most common protozoa were T. muris 27.0%, followed by G. muris (9.3%) and E. muris (6.9%). T. muris was the most common protozoa (8,17,18). Overall prevalence of intestinal parasites in rat in Arbil was 76% including T. muris with the higher incidence of 56%, G. muris 12%, H. muris 8% and the least infection percentage was 4% for E. muris (17). The result of our study is consistent with that.
Prevalence of protozoa infections have been reported in mice and rat, Entamoeba sp.  (19). Prevalence rate of protozoa infection in our study was higher than those have been reported from North America and Europe.
Encephalitozoon sp. has been reported from rabbits, mice, guinea pigs and rats. The organisms are small, bipolar and rod-like. They occur singly or in clumps (4). Three strains (I, II, and III) are recognized in E. cuniculi, which, according to the host of the originally characterized isolates, are also designated "rabbit strain," "mouse strain" and "dog strain" (21).
In the present study, microspora spores were identified in 63(31.03%) of all samples. Encephalitozoon sp. was found in (55%) of mice samples by parasitological method (22).
In our study from coccidia group: with the higher incidence of Eimeria spp 46(22.7%), followed by Isospora spp 4(2%) and 1(0.5%) Cryptosporidium spp were found. In concentrated pellets, some unsporulated oocysts were found and after cultivation in dichromate potassium and observation of pattern of four sporocysts and two sporozoites, Eimeria spp diagnosis was made and about Isospora unsporulated and sporulated oocysts were seen. Eimeria is the most parasites from coccidian group in rodents in our study and it was also described in some previous study (4,23,24).The overall prevalence of Eimeria spp in rabbits from pet shops and farms were 46.2% and 41.7%, respectively in Taiwan that is similar our result (23,24).
C. muris has been reported from mice and the guinea pig. They are true coccidia whose developmental stages appear to take place on the surface of the host cell but not within the cell proper. In the case of C. muris, the parasite may be seen in large numbers in sections of the stomach and is a parasite of the peptic glands, but C. parvum has been reported in rat and found in the glandular structures of the small intestine of the mouse (4,25). In this study, one Cryptosporidium spp positive was de-tected in a sample belongs to a M. persicus. Molecular investigation is needed for confirmation of Cryptosporidium species.
Protozoa infection in male rodents 69(57%) was more than female 52(43%). In the present study, there was no significant difference between male and female rodents in consistent with another others (26).

Conclusion
Rodents as reservoirs of some important parasites in this area infected with some zoonotic parasites, hence control of these animals has an important role on prevention of public health problems.